The progression of a cell through the mitotic cycle is controlled by an array of proteins. Central among them is p34.sup.cdc2, a serine/threonine protein kinase that determines the induction of a variety of specific mitotic events (Murray and Kirschner, 1989; Nurse, 1990; Draetta, 1990). The p34.sup.cdc2 kinase, or a closely related variant, is also involved in the induction of DNA replication in S phase (Blow and Nurse, 1990; D'Urso et al., 1990; Fang and Newport, 1991). The activity of p34.sup.cdc2 is in turn controlled by a series of specific phosphorylation and dephosphorylation events on p34.sup.cdc2 (Nurse, 1990), and by association of p34.sup.cdc2 with various cyclins (Minshull et al., 1989; Reed, 1991), proteins whose abundance oscillates as the cell cycle advances.
The progression of the cell to the next stage of its cycle is under the control of factors that act as "checkpoints" which assure that the previous stage has been completed before the subsequent stage ensues (Hartwell and Weinert, 1989). The cell contains exquisitely sensitive feedback control circuits that can, for example, prevent exit from S phase if a fraction of a percent of DNA remains unreplicated (Dasso and Newport, 1990), and can block advance into anaphase in mitosis until all the chromosomes have aligned on the metaphase plate (Rieder and Alexander, 1990). The nature of these checkpoints, and how they act to block cell cycle progression, is unknown.
Various mutants have been isolated which escape specific cell cycle control circuits and progress inappropriately to the next cell cycle stage. They include wee1 mik1 double mutants (Lundgren et al., 1991), pim1 (Matsumoto and Beach, 1991), and rad9 (Weinert and Hartwell, 1988) in yeast, bimE7 in Aspergillis (Osmani et al., 1988), and the RCC1 mutant tsBN2 in mammalian BHK cells (Nishimoto et al., 1978). All of these mutants exhibit an uncoupling of entry into mitosis from the completion of DNA replication. In addition, drug treatments such as the combination of exposure to the DNA replication inhibitor hydroxyurea with exposure to caffeine can cause normal mammalian cells to enter mitosis without completing S phase (Schlegel and Pardee, 1986). Recently, mutations in S. cerevisiae, bub (Hoyt and Roberts, 1991) and mad (Li and Murray, 1991), have been isolated which fail to arrest in mitosis with microtubial destabilizing drugs.
The purine analogue 2-aminopurine (2-AP), a specific protein kinase inhibitor (Farrell et al., 1977; Mahadevan et al., 1990), has been shown to cause S-phase arrested cells to inappropriately enter mitosis (Schlegel et al., 1990).
We have now found that 2-aminopurine (2-AP) also causes BHK cells in mitotic arrest to rapidly exit mitosis. As the drug has the capacity to advance cells inappropriately past checkpoints at two distinct parts of the cell cycle, this result indicated that there might be an underlying common factor responsible for the various inhibitory controls of the cell cycle. We have therefore tested the capacity of 2-AP to inappropriately advance the cell cycle following cell blockage with a variety of stage specific inhibitors. We here report the striking result that 2-AP causes cells to override every cell cycle block point examined, regardless of whether the arrest point is in G.sub.1, S phase, G.sub.2, or mitosis. Further, it appears that cells exposed continuously to 2-AP may exit S phase without completion of replication, and may exit mitosis without metaphase, anaphase, or telophase events. Of the various cell cycle checkpoints, only one has thus far been associated with a specific molecular event. The capacity of a cell to progress from G.sub.2 into mitosis is controlled by the state of phosphorylation of p34.sup.cdc2 on a tyrosine residue (Gould and Nurse, 1989). As this p34.sup.cdc2 phosphorylation state is specific to the G.sub.2 stage of the cell cycle (Gould and Nurse, 1989), there does not appear to be a link between this inhibitory event and the checkpoints to cell cycle progression that occur in other stages of the cell cycle. Nonetheless, we now have cause to believe an underlying commonality exists, perhaps at the level of a specific 2-AP sensitive protein kinase.
Several of the inhibitors that we have used to induce cell cycle arrest (hydroxyurea, VM-26, and taxol) are used therapeutically for cancer treatment (O'Dwyer et al., 1984; Rowinsky et al., 1990). Neither drug of itself is lethal to culture cells during short exposure. However, inappropriate exit from an arrested state, induced by 2-AP, is ultimately lethal for the cell. Therefore, our results suggest that "binary" therapy, using a drug such as VM-26 or taxol in combination with 2-AP or another such purine analogue will cause inappropriate escape from cell cycle blockage, with a synergistic destructive effect on tumors.